Our general research interest is the understanding of the assembly of macro-molecules. This proposal involves the study of the sequence of events occuring in vivo in the association of alkaline phosphatase (APase) with the membrane of Bacillus licheniformis MC14 and the study of the in vitro reconstitution of the APase and purified membranes. The in vivo study involves the monitoring of APase protein (subunits or oligomer) in the cytosol, membrane, and culturing medium during growth under derepressed conditions. In an effort to observe a system where protein synthesis is continuing but cell membrane synthesis is stopped we propose to incubate spheroplasts in a low phosphate medium containing C14 amino acids and monitor as above. In both in vivo systems enzymatic and immunological assays will be used. The in vitro study involves understanding the solubilizations which we have affected, preparing membranes suitable for reconstitution, determining the in vivo location of the APase and reconstituting the system. A number of solubilizations have been developed which yield a homogeneous product with respect to size on sucrose gradients in the presence of the agent but only one method yields a product which is soluble in aqueous solution after removal of the solubilizing agent. This method involves an 80 degrees C heat step in the presence of l M Mg ions. The enzyme isolated from this solubilization has been characterized. We propose to isolate and compare the Mg ions dependent solubilized enzyme to determine what changes have occurred during the rather drastic heat step which results in a truly soluble enzyme. We plan a thorough study of the localization of the enzyme to gain information which will be used in assessing the similarity between in vivo assembly and the reconstitutional assembly.